Hypermax films

For protein expression analyses, cells from different blood donors were pooled to obtain sufficient amounts of protein. The whole-cell lysates from cell lines or pooled primary cells were prepared in the passive lysis buffer of the dual luciferase assay kit (Promega) containing 10 mM Na₃PO₄. Equal amounts of protein (10 to 30 μg/lane) were separated by SDS-PAGE and transferred to Hybond-P polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences). The membranes were blocked with 5% milk protein in PBS. Antibodies against IRF3 and MxA were as previously described (74 (link), 75 (link)), and antibodies against SARS-CoV N protein and SARS-CoV-2 S1 protein were prepared as described above. Staining was done in blocking buffer at room temperature for 1 h. Antibodies against phosphorylated IRF3 (P-IRF3; 4947), p38 (9212), phosphorylated p38 (P-p38; 9211L), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 2118) were from Cell Signaling Technology, and staining was done in Tris-buffered saline, pH 7.4, containing 5% bovine serum albumin (BSA) at 4°C overnight. Horseradish peroxidase (HRP)-conjugated antibodies (Dako) were used in the secondary staining at room temperature for 1 h. Protein bands were visualized on HyperMax films using an ECL plus system (GE Healthcare).

For protein expression analyses, cells from different blood donors were pooled to obtain sufficient amounts of protein. The whole-cell lysates from cell lines or pooled primary cells were prepared in the passive lysis buffer of the dual luciferase assay kit (Promega) containing 10 mM Na₃PO₄. Equal amounts of protein (10 to 30 μg/lane) were separated by SDS-PAGE and transferred to Hybond-P polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences). The membranes were blocked with 5% milk protein in PBS. Antibodies against IRF3 and MxA were as previously described (74 (link), 75 (link)), and antibodies against SARS-CoV N protein and SARS-CoV-2 S1 protein were prepared as described above. Staining was done in blocking buffer at room temperature for 1 h. Antibodies against phosphorylated IRF3 (P-IRF3; 4947), p38 (9212), phosphorylated p38 (P-p38; 9211L), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 2118) were from Cell Signaling Technology, and staining was done in Tris-buffered saline, pH 7.4, containing 5% bovine serum albumin (BSA) at 4°C overnight. Horseradish peroxidase (HRP)-conjugated antibodies (Dako) were used in the secondary staining at room temperature for 1 h. Protein bands were visualized on HyperMax films using an ECL plus system (GE Healthcare).